This invention relates to analytical tools and methods for assaying biological materials in a variety of applications. In particular, the invention relates to an apparatus, systems and a method for assaying biological materials for monitoring levels of gene expression and mutations in gene sequences using an annular format.
Conventional analysis of biological materials, such as DNA, RNA, proteins and the like, employs an apparatus having biological material on a substrate in an array pattern of discrete features. The features are typically chemically bound to the substrate. The features may be either xe2x80x9cprobesxe2x80x9d of known molecular make-up or xe2x80x9ctargetsxe2x80x9d of unknown molecular make-up. For the purposes of simplicity, hereinafter the features bound to the substrate will be referred to as probes and the samples under test will be referred to as xe2x80x9ctargetsxe2x80x9d. Arrays of biological probes are quickly becoming a powerful method of simultaneously assaying thousands of targets within a single biological sample. The surface bound probes are typically formed of DNA oligonucleotides, cDNA""s, PCR products, antibodies, antigens, and the like.
The arrays are manufactured using automated equipment, such that the spatial location on the substrate of each type of surface bound probe is known within a certain margin of error. The sample containing the unknown quantities of the targets is modified so that each potential target molecule is labeled with a fluorescent label. The sample is applied to the array surface so that the targets may hybridize or bind to their complementary surface bound probe. After the reaction is complete, the surface of the array is washed. The hybridized array is interrogated optically to determine the level of hybridization and the locations and therefore, the identity of the hybridized targets. Optionally, the array substrate may be put into a package for handling, processing and optical interrogation. The array can be held in the package with an appropriate adhesive or glue.
Optical interrogation is typically performed with commercially available optical scanning systems, examples of which are described in U.S. Pat. Nos. 5,837,475, 5,760,951 (confocal scanner) and U.S. Pat. No. 5,585,639 (off axis scanner), all incorporated herein by reference. Typical scanning fluorometers are commercially available from different sources, such as Molecular Dynamics of Sunnyvale, Calif., General Scanning of Watertown, Mass., Hewlett Packard of Palo Alto, Calif. and Hitachi USA of So. San Francisco, Calif. Analysis of the data, (i.e., collection, reconstruction of image, comparison and interpretation of data) is performed with associated computer systems and commercially available software, such as IMAGEQUANT(trademark) by Molecular Dynamics or GENECHIP(trademark) by Affymetrix of Santa Clara, Calif. Typically, a laser beam is scanned across the array surface. The laser beam excites the fluorescent labels on the hybridized targets and the fluorescent signal is detected by a detector and processed by a computer. The intensity of the signal at each physical location in the array is a measure of the hybridization efficiency of a target with a known chemical probe. The intensity relates directly to the concentration of that target within the sample. The signal can be used to simply identify the targets within the unknown or quantitate the targets. The identity of the target is known since it is the complement of the probe.
While there are many methods known in the art for forming and analyzing such arrays, all of them assume that the array will be created and, subsequently analyzed or xe2x80x9creadxe2x80x9d in xe2x80x9can x, y formatxe2x80x9d, where xe2x80x9cxxe2x80x9d and xe2x80x9cyxe2x80x9d are the coordinate axes of a two dimensional Cartesian coordinate system. xe2x80x9cReadingxe2x80x9d an array in an x, y format typically requires raster scanning a laser beam across the array surface. Typically, either the laser beam and the fluorescence detection system must be moved or the array must be moved in relation to the optics to scan in a raster pattern or x, y format. In either case, the system typically requires some kind of x, y table or a single axis motion table and a galvanometer.
Typical specifications of a biological array require a very sensitive optical assay. Therefore, the size of the laser beam is normally focussed to about 5 to 10 microns to meet the sensitivity requirements of the assay. In order to scan an array surface in increments of 5 or 10 microns, the x, y table or galvanometer must be very precise. Such precision is normally expensive. The price of an optical scanner is typically above $50,000, which can be cost prohibitive for small analytical laboratories.
Moreover, if an x, y table is employed to move either the optics or the array in a raster scan fashion, the x, y table must be moved in a precise way. In particular, the table must be moved: (i) very quickly in one direction; (ii) it must accelerate up to xe2x80x9creading velocityxe2x80x9d prior to the beam touching the array; (iii) then it must move across the array at a constant speed; (iv) decelerate outside of the array area; (v) reverse direction; and then repeat steps (i)-(v) until the surface is completely scanned. A significant portion of the total time required to scan the array is spent in making changes in the direction during the raster scan.
Moreover, in the x, y format, the laser beam must be scanned beyond the region of interest on the array to allow space for the deceleration of the beam and for the beam to reverse direction. Since the array under test is normally glued into a package, the beam may hit a glue edge or line. Conventionally, adhesive glues are often fluorescent and are likely to be more fluorescent than the signals from the very sensitive array. If the laser beam encounters the glue line, the resulting signal can overload the detection system. Overload can result in either temporary degradation of the detection channel or permanent damage.
If a galvanometer is employed, the collection optics must be much larger to collect signal from the entire swept line. Larger optics are more expensive and more likely to have aberrations.
Therefore, it would be advantageous to have an assay system that does not require the complexities and expense of, and avoids the problems associated with, the traditional x, y raster scan format.
U.S. Pat. No. 5,508,200 Tiffany et al., discloses an automated chemical analysis system for high volume chemical testing that creates arrays of chemical reactions on an absorbent media or a solid substrate patterned with microwells. The system includes a dispensing mechanism for dispensing multiple reagents or test samples within a common test area on the media. The common test area has microcuvettes or microwells to hold the samples and reagents and prevent commingling of chemicals between each sample. Mixing of the samples with the reagents is either performed before dispensing or on the media. Tiffany employs a CCD camera to simultaneously monitor the individual reactions at the discrete locations within the common area.
In one embodiment, Tiffany discloses using a rotatable circular disk of an absorbent matrix instead of a continuous strip for the purpose of accommodating multiple sample spots. As a result, photometric measurements with the CCD camera can be made at a single station. The circular disk is rotated to bring the set of microcuvettes into position for photometric measurement. The circular disk format facilitates analyses requiring measurement at variable time intervals and also multipoint rate analyses because of the ease of returning to the single camera station.
However for several reasons, the chemical analysis system described by Tiffany et al. is not conducive to the conventional analysis of biological materials using arrays, as described above. The arrays of biological materials require that each feature be exposed to the entire quantity of the target sample since the concentration of the analytes is typically orders of magnitude less than that of chemistries Tiffany et al. use. The analysis system of Tiffany et al. requires fluid isolation between each location. This is achieved by physical isolation (microwells) or by using such small fluid aliquots on absorbent media that the fluid spots do not connect.
In the manufacture of conventional biological arrays, the read probes are chemically bound to the substrate and the targets are hybridized to the read probes. Tiffany et al. disclose using reagents and samples that are dried onto the absorbent media or captured within a microwell. Therefore, the system of Tiffany et al. would not be conducive to the conventional hybridization and subsequent wash steps.
Moreover, Tiffany et al. disclose using CCD cameras for optical interrogation of the individual chemical reactions. Current technology in CCD cameras is not as sensitive to fluorescent signal as a laser beam with a photomultiplier tube detector used in conventional biological array assays. The conventional biological array assay described above typically scans the entire surface in a systematic pattern rather than taking simultaneous camera shots of an entire area.
The methodologies disclosed by Tiffany et al. dictate a xe2x80x9cdispense and immediately readxe2x80x9d process. These methodologies are not useful to conventional biological array assays, which require reaction times of at least 30 minutes to overnight to complete. Furthermore, Tiffany et al. disclose using homogeneous chemistries in the chemical analysis system. The conventional biological array allows for heterogeneous assays.
U.S. Pat. No. 4,940,322 Miwa et al. discloses a fluorescent analysis apparatus for measuring the presence of microorganisms using fluorescent substances. Each sample is placed in a discrete container with reactive liquids. The containers are placed in a rotatable holder or carousel in a reaction section of the system. After a specified time, the containers are rotated into position by means of a motor and gearing arrangement at a measuring section of the system, where they are interrogated with a fluorescent excitation light.
The analysis apparatus disclosed by Miwa et al. is also not conducive to biological array assays for many of the same reasons mentioned above for Tiffany et al. In particular, the apparatus of Miwa et al. does not allow for conventional manufacture and hybridization processes used in conventional biological array assays.
U.S. Pat. No. 5,812,272 issued to King et al. and assigned to the assignee of the present invention, discloses an apparatus and method for analyzing target chemicals with a tiled light source array. At column 9, line 28, it is disclosed that the light source tiles can be arranged in a circular pattern and held in place by grooved channels formed within a support. However, there is no disclosure of why or how the circular pattern of tiles is used and if there are any advantages to using a circular pattern of light source tiles.
Thus, it would be advantageous to have an apparatus and method for assaying biological materials that follows much of the conventional array manufacturing and analysis methodologies, but employs a less complex interrogation scheme.
The present invention provides an apparatus, systems and methods for assaying biological materials which use an r, xcex8 format, where xe2x80x9crxe2x80x9d and xe2x80x9cxcex8xe2x80x9d are the coordinate axes of a two dimensional polar coordinate system. In accordance with the present invention, the complexities and expense of conventional optical interrogation equipment and methods are overcome, without compromising assay sensitivity, precision and speed.
The apparatus of the present invention is an array of discrete features of biological material in a circular or annular array pattern. The present array is manufactured using conventional materials, manufacturing equipment and methods, except that the biological materials are deposited onto a substrate in the circular or annular array pattern.
One system of the present invention is a system for synthesizing arrays that includes a spinner assembly for rotating the apparatus during the synthesis and deposition of the biological material on the substrate to form the circular or annular pattern of biological features. The spinner assembly provides efficient means for annular deposition and spreading and removing ancillary materials used in the synthesis process.
Another system of the present invention is a system for hybridizing the array apparatus of the invention with a complementary biological material. The hybridization system uses well-established methods and materials for hybridization. However, the hybridization system further includes a spinner assembly to rotate the apparatus after the complementary biological material is added. The spinning motion spreads the complementary biological material efficiently over the annular array such that less complementary biological material is needed for the assay. Further the spinning motion effectively removes unhybridized material and ancillary materials and moves any bubbles that form out of the array area. In the systems of synthesizing and hybridizing, the spinner assemblies are used to spin the array such that the advantages of centrifugal force on array processing are realized.
Still another system of the present invention is a system for optically interrogating the apparatus of the present invention. The interrogation system includes a conventional light source to emit a light beam, optics, detection and analysis subsystems. Moreover, the interrogation system of the present invention employs a scanning assembly that holds and rotates the apparatus so that the light source and optics remain stationary, and a galvanometer is not necessary. The hybridized apparatus is scanned and read in a r, xcex8 format using a spinner subassembly. The optical interrogation further comprises a linear stage to move the apparatus or the optics radially to efficiently expose all features in the annular array pattern on the apparatus to the light source.
The r, xcex8 format is simpler to use and implement than the conventional x, y format. Detection of the fluorescent signals from the hybridized targets is accomplished with detection systems employing conventional photomultiplier tubes, so that sensitivity is not compromised. The signal information gathered by the conventional detection system is stored and processed by conventional analysis systems that are adapted to process r, xcex8 formatted data and provide information about the target sample under investigation.
In accordance with the invention, the optics in the optical scanner advantageously remain focused on a single point. The array is scanned in a spiral pattern or by sweeping around a circle at a specific radius and then stepping to a next radius and sweeping in a circle again. The scan profile is scanned either with a constant angular and radial speed or with a constant rate of area scanned. The entire scan time is dedicated to reading active array rather than accelerating and decelerating after every scan line, as in the conventional x, y format. The present system requires a motor to rotate the array and a linear system to move the array radially. In another embodiment, the linear system is adapted to move the read optics instead of the array. Therefore, the present invention avoids the expense of a precision x, y table in the optical scanning equipment.
The present system allows the optics to be smaller, lower weight and less expensive. Optics with small field of view can be made with high light collection efficiency (high numerical aperture) since aberrations are more easily avoided. A xe2x80x9chighxe2x80x9d numerical aperture starts at approximately 0.5-0.6. The optics only need to be corrected for a field of view covering the alignment tolerances, as in conventional systems. In addition, the present system avoids using large galvanometer mirrors, which are otherwise associated with high numerical aperture scanners.
Moreover, in accordance with the invention, the present system avoids the risk of detector overload found in the optical scanners using the x, y format discussed above. The array may be packaged in some applications. Advantageously, the array is adhered using an annular glue line, rendering the glue line concentric with the scan pattern. Therefore, there is no risk of the laser beam crossing the glue line and overloading the detection system.
The present invention also includes a method for assaying biological materials. The method includes the steps of providing an array of biological material on a substrate in an annular array of discrete features, hybridizing the array with another biological material, optically interrogating the hybridized array and determining information about the biological materials from the results of the interrogation. The steps of the method use the respective systems for synthesizisg, hybridizing and optically interrogating the apparatus. The method takes advantage of the use of the spinner assemblies to implement the r,xcex8 format in accordance with the invention. Rotating and spinning the substrate in the synthesis, the hybridization and the optical interrogation of the array provide many advantages to the process of assaying biological materials that are not found in conventional systems using an x, y format
The present invention provides additional advantages. For example, during the manufacture of the hybridized array apparatus, centrifugal force is applied to the fluids on the array. Quite advantageously, the centrifugal force simplifies the washing procedure after hybridization because the fluids are easily removed by spinning the substrate. Moreover, the centrifugal force advantageously minimizes the quantity of target sample required for analysis in several ways: First, the target sample is spread efficiently over the array features by the spinning motion, so that little target sample is needed for hybridization. In particular, in a preferred embodiment, the apparatus is packaged in a housing, where little target sample is needed to fill the fluid channel in the package leading to the array surface. Second, the surface energy of the package array requires less control since capillary action is not needed to fill the fluid channel in the package. Third, the filling and emptying of the fluid cavity in the package can be handled automatically. Moreover, any bubbles in the fluid, which form during the sample loading or hybridization steps, are advantageously spun out of the active read area.